dissection-culture-commissural-neurons-from-embryonic-spinal

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival.

An essential role for DNA methyltransferase DNMT3B in cancer cell survival.

Eine wesentliche Rolle f&#xFC;r die DNA-Methyltransferase DNMT3B in das &#xDC;berleben von Krebszellen.

Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2'-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2'-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2'-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells.

DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells.

DNMT1 ist erforderlich, um CpG-Methylierung und aberrante Gen-Silencing in menschlichen Krebszellen zu erhalten.

HAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis.

MEF2-dependent recruitment of the HAND1 transcription factor results in synergistic activation of target promoters.

MEF2-abh&#xE4;ngige Rekrutierung der HAND1 Transkriptionsfaktor f&#xFC;hrt zu synergistischen Aktivierung des Ziel-Promotoren.

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.

Boc is a receptor for sonic hedgehog in the guidance of commissural axons.

Im R&uuml;ckenmark ist sonic Hedgehog (Shh) von der Bodenplatte zur Steuerung der Generation unterschiedliche Klassen von ventralen Neuronen entlang der dorsoventral ausgeschieden. Genetische und in-vitro-Studien haben gezeigt, Shh auch sp&auml;ter fungiert als eine Mittellinie abgeleitete Chemoattractant f&uuml;r kommissurale Axone. Die Receptor(s) f&uuml;r Shh Attraktion bleiben jedoch unbekannt. Hier zeigen wir, dass zwei Robo-&auml;hnliche Proteine, Boc und Cdon, speziell an Shh binden und damit Kandidaten Rezeptoren f&uuml;r die Aktion der Shh als ein Axon-Anleitung-Ligand. BOC wird ausgedr&uuml;ckt durch kommissurale Neuronen und gezielte Unterbrechung der Boc in Maus f&uuml;hrt die Irref&uuml;hrung der kommissurale Axone in Richtung der Bodenplatte. RNA-Interferenz-vermittelter Niederschlag von Boc beeintr&auml;chtigt die F&auml;higkeit der Ratte kommissurale Axone, sich eine ektopische Quelle von Shh in-vitro-zuzuwenden. Zusammengenommen lassen diese Daten Boc notwendig als ein Rezeptor f&uuml;r Shh in kommissurale Axon Anleitung.

BOC ist ein Rezeptor f&uuml;r sonic Igel in der Leitung von kommissurale Axone.

[Sonic Hedgehog und Boc: eine sehr attraktive Geschichte &uuml;ber Axone].

[Sonic hedgehog and Boc: a very attractive story about axons].

Developing axons are attracted to the CNS midline by Netrin proteins and other as yet unidentified signals. Netrin signals are transduced in part by Frazzled (Fra)/DCC receptors. Genetic analysis in Drosophila indicates that additional unidentified receptors are needed to mediate the attractive response to Netrin. Analysis of Bolwig's nerve reveals that Netrin mutants have a similar phenotype to Down Syndrome Cell Adhesion Molecule (Dscam) mutants. Netrin and Dscam mutants display dose sensitive interactions, suggesting that Dscam could act as a Netrin receptor. We show using cell overlay assays that Netrin binds to fly and vertebrate Dscam, and that Dscam binds Netrin with the same affinity as DCC. At the CNS midline, we find that Dscam and its paralog Dscam3 act redundantly to promote midline crossing. Simultaneous genetic knockout of the two Dscam genes and the Netrin receptor fra produces a midline crossing defect that is stronger than the removal of Netrin proteins, suggesting that Dscam proteins also function in a pathway parallel to Netrins. Additionally, overexpression of Dscam in axons that do not normally cross the midline is able to induce ectopic midline crossing, consistent with an attractive receptor function. Our results support the model that Dscam proteins function as attractive receptors for Netrin and also act in parallel to Frazzled/DCC. Furthermore, the results suggest that Dscam proteins have the ability to respond to multiple ligands and act as receptors for an unidentified midline attractive cue. These functions in axon guidance have implications for the pathogenesis of Down Syndrome.

Dscam guides embryonic axons by Netrin-dependent and -independent functions.

Entwickelnden Axone sind zu der CNS-Mittellinie von Netrin Proteine und andere bisher nicht identifizierten Signale angezogen. Netrin-Signale sind teilweise durch Frazzled (Fra) transduzierten / DCC-Rezeptoren. Genetische Analyse in Drosophila zeigt, dass zus&auml;tzliche unidentifizierte Rezeptoren erforderlich sind, um die attraktive Antwort auf Netrin zu vermitteln. Analyse der Bolwigs Nerven zeigt, dass Netrin-Mutanten einen &auml;hnlichen Ph&auml;notyp, Down-Syndrom Zelladh&auml;sionsmolek&uuml;l (Dscam)-Mutanten haben. Netrin und Dscam Mutanten Display Dosis sensible Interaktionen, was darauf hindeutet, dass Dscam als ein Netrin-Rezeptor handeln k&ouml;nnte. Wir zeigen mit der Zelle-Overlay-Assays, die Netrin bindet um zu fliegen und vertebrate Dscam und, dass Dscam mit der gleichen Affinit&auml;t wie DCC Netrin bindet. Bei der CNS-Mittellinie finden wir, dass dieser Dscam und seine Paralog Dscam3 redundant handeln, um die Mittellinie &uuml;berschreiten zu f&ouml;rdern. Gleichzeitige genetische k.o. von den zwei Dscam-Genen und dem Netrin-Rezeptor Fra produziert einen Mittellinie-Kreuzung-Mangel, der ist st&auml;rker als die Entfernung von Netrin Proteine, darauf hindeutet, dass die Dscam Proteine auch in einen Weg parallel zur Netrins fungieren. Dar&uuml;ber hinaus kann eine &Uuml;berexpression von Dscam in Axone, die normalerweise nicht die Mittellinie &uuml;berqueren ektopische Mittellinie &uuml;berschreiten, mit einem attraktiven Rezeptor-Funktion zu induzieren. Unsere Ergebnisse unterst&uuml;tzen das Modell, das Dscam Proteine als attraktive Rezeptoren f&uuml;r Netrin-Funktion und auch parallel zur Frazzled/DCC. Die Ergebnissen zufolge dar&uuml;ber hinaus Dscam Proteine haben die M&ouml;glichkeit, mehrere Liganden reagieren und agieren als Rezeptoren f&uuml;r ein nicht identifizierter Mittellinie attraktive Cue. Diese Funktionen in Axon Anleitung haben Auswirkungen auf die Pathogenese des Down-Syndroms.

Dscam f&uuml;hrt die embryonalen Axone von Netrin-abh&auml;ngige und -unabh&auml;ngige Funktionen.

Sonic hedgehog (Shh) plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, we developed an in vitro assay for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, we showed that attraction by Shh does not require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons.

Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway.

Sonic Hedgehog (Shh) spielt wesentliche Rollen in entwicklungspolitischen Veranstaltungen wie Zelle Schicksal Anleitung zur Spezifikation und Axon. SHH induziert Zelle Schicksal Spezifikation durch kanonische Shh Signalisierung, vermittelt durch Transkription. Der Mechanismus, mit dem Shh Axone f&uuml;hrt, ist jedoch unbekannt. Um dies zu untersuchen, haben wir ein in vitro Tests Axon-Anleitungen, in denen Neuronen abgebildet werden k&ouml;nnen, w&auml;hrend die Reaktion auf einen definierten Farbverlauf von einer chemischen Cue entwickelt. Axone der dissoziiert kommissurale Neurons in einem Shh Farbverlauf drehte die rasch zunehmende Konzentrationen von Shh. im Einklang mit dieser Sofortreaktion platziert, wir zeigten, dass die Anziehung von Shh Transkription nicht erforderlich ist. Stattdessen regt Shh die Aktivit&auml;t der Src Familie Kinase (SFK) Mitglieder in gewissem Sinne gegl&auml;ttet-abh&auml;ngigen. Dar&uuml;ber hinaus ist die SFK Aktivit&auml;t erforderlich, f&uuml;r die Ausrichtung der Shh-vermittelte der kommissurale Axone, aber nicht f&uuml;r Induktion Gli Reporter transkriptionelle Aktivit&auml;t. Zusammen, zeigen diese Ergebnisse, dass Shh &uuml;ber eine rasch handeln, Mitgliedattribut Signalweg um Axone leiten fungiert.

Sonic Hedgehog f&uuml;hrt Axone durch eine Mitgliedattribut, Src-Familie-Kinase-abh&auml;ngigen Signalweg.

Mirror movements are involuntary contralateral movements that mirror voluntary ones and are often associated with defects in midline crossing of the developing central nervous system. We studied two large families, one French Canadian and one Iranian, in which isolated congenital mirror movements were inherited as an autosomal dominant trait. We found that affected individuals carried protein-truncating mutations in DCC (deleted in colorectal carcinoma), a gene on chromosome 18q21.2 that encodes a receptor for netrin-1, a diffusible protein that helps guide axon growth across the midline. Functional analysis of the mutant DCC protein from the French Canadian family revealed a defect in netrin-1 binding. Thus, DCC has an important role in lateralization of the human nervous system.

Mutations in DCC cause congenital mirror movements.

Spiegel-Bewegungen sind unfreiwillige kontralaterale Bewegungen, die freiwillige diejenigen zu spiegeln und sind oft verbunden mit defekten Mittellinie &Uuml;berquerung der entwickelnden zentralen Nervensystems. Wir studierten zwei gro&szlig;e Familien, ein franz&ouml;sisch-kanadischer und eine iranische, in denen isolierte kongenitale Spiegel Bewegungen, autosomal-dominant vererbt geerbt wurden. Wir fanden, dass die betroffenen Protein-Abschneiden Mutationen in DCC (gel&ouml;schte in kolorektales Karzinom), ein Gen auf Chromosom 18q21.2, die einen Rezeptor f&uuml;r Netrin-1, ein diffusible Protein kodiert durchgef&uuml;hrt, dass hilft Axon Wachstum &uuml;ber der Mittellinie Wegweiser. Funktionsanalyse von den Mutanten DCC Protein aus der Familie der frankokanadische ergab einen Defekt im Netrin-1 Bindung. DCC hat somit eine wichtige Rolle in Lateralisations des menschlichen Nervensystems.

Mutationen im DCC f&uuml;hren angeborene Spiegel Bewegungen.

Hedgehog (Hh) proteins regulate important developmental processes, including cell proliferation and differentiation. Although Patched acts as the main Hh receptor in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, an Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules-Boc, Cdon, and Gas1-have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand that binds Patched1 but not Boc, Cdon, or Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.

Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation.

Igel (Hh) Proteine regulieren wichtige Entwicklungsprozesse, einschlie&szlig;lich der Zellproliferation und Differenzierung. Obwohl flickte als den wichtigsten Hh-Rezeptor in Drosophila dient, erfordert Hh Signalisierung absolut die zus&auml;tzliche Hh-bindene Proteine Ihog und Boi. Hier zeigen wir, dass vermehren unerwartet, zerebell&auml;re Untermodul Neuron Vorl&auml;uferzellen (CGNPs) fehlende Boc und Cdon, die Vertebraten Orthologen Ihog und Boi, noch als Reaktion auf Hh. Dies ist, weil in Ermangelung Hh Signalisierung Gas1, ein Hh-verbindliches Protein nicht vorhanden in Drosophila, vermittelt. Konsequent, nur CGNPs fehlen alle drei Molek&uuml;le-Boc, Cdon und Gas1-haben ein vollst&auml;ndiger Verlust der Hh-abh&auml;ngige Verbreitung. In komplement&auml;rer Weise finden wir, dass ein mutierter Hh-Ligand, die Patched1 aber nicht Boc, Cdon oder Gas1 bindet Hh Signalisierung nicht aktivieren kann. Zusammen, das demonstriert eine unbedingte Voraussetzung f&uuml;r Boc, Cdon und Gas1 in Hh Signalisierung und zeigt unterschiedliche Basis f&uuml;r Ligand-Bindung-Komponenten, die die Vertebraten und Wirbellosen Hh-Rezeptor-Systemen unterscheidet.

Article	Year
An essential role for DNA methyltransferase DNMT3B in cancer cell survival. The Journal of biological chemistry\| PubMed ID: 12015329	2002
DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nature genetics\| PubMed ID: 12496760	2003
MEF2-dependent recruitment of the HAND1 transcription factor results in synergistic activation of target promoters. The Journal of biological chemistry\| PubMed ID: 16043483	2005
Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature\| PubMed ID: 17086203	2006
[Sonic hedgehog and Boc: a very attractive story about axons]. MÃ©decine sciences : M/S\| PubMed ID: 18272068	2008
Dscam guides embryonic axons by Netrin-dependent and -independent functions. Development (Cambridge, England)\| PubMed ID: 18948420	2008
Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway. Neuron\| PubMed ID: 19447091	2009
Mutations in DCC cause congenital mirror movements. Science (New York, N.Y.)\| PubMed ID: 20431009	2010
Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Developmental cell\| PubMed ID: 21664577	2011

Steves Morin

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